Oocyst Shedding Dynamics in Children with Cryptosporidiosis: a Prospective Clinical Case Series in Ethiopia

ABSTRACT Knowledge on the duration of Cryptosporidium oocyst shedding, and how shedding may be affected by subtypes and clinical parameters, is limited. Reduced transmission may be a secondary benefit of cryptosporidiosis treatment in high-prevalence areas. We conducted a prospective clinical case series in children of <5 years presenting with diarrhea to a health center and a hospital in Ethiopia over an 18-month period. Stool samples were collected repeatedly from children diagnosed with cryptosporidiosis for up to 60 days. Samples were examined, and Cryptosporidium shedding was quantified, using auramine phenol, immunofluorescent antibody staining, and quantitative PCR (qPCR). In addition, species determination and subtyping were used to attempt to distinguish between new infections and ongoing shedding. Duration and quantity of shedding over time were estimated by time-to-event and quantitative models (sex- and age-adjusted). We also explored how diarrheal severity, acute malnutrition, and Cryptosporidium subtypes correlated with temporal shedding patterns. From 53 confirmed cryptosporidiosis cases, a median of 4 (range 1 to 5) follow-up stool samples were collected and tested for Cryptosporidium. The median duration of oocyst shedding was 31 days (95% confidence interval [CI], 26 to 36 days) after onset of diarrhea, with similar estimates from the quantitative models (31 days, 95% CI 27 to 37 days). Genotype shift occurred in 5 cases (9%). A 10-fold drop in quantity occurred per week for the first 4 weeks. Prolonged oocyst shedding is common in a pediatric clinical population with cryptosporidiosis. We suggest that future intervention trials should evaluate both clinical efficacy and total parasite shedding duration as trial endpoints. IMPORTANCE Cryptosporidiosis is an important cause of diarrhea, malnutrition, and deaths in young children in low-income countries. The infection spreads from person to person. After infection, prolonged release of the Cryptosporidium parasite in stool (shedding) may contribute to further spread of the disease. If diagnosis and treatment are made available, diarrhea will be treated and deaths will be reduced. An added benefit may be to reduce transmission to others. However, shedding duration and its characteristics in children is not well known. We therefore investigated the duration of shedding in a group of young children who sought health care for diarrhea in a hospital and health center in Ethiopia. The study followed 53 children with cryptosporidiosis for 2 months. We found that, on average, children released the parasite for 31 days after the diarrhea episode started. Point-of-care treatment of cryptosporidiosis may therefore reduce onward spread of the Cryptosporidium parasite within communities and households.

Cryptosporidium spp. in children under 5 years of age with diarrhoea in a low-income country (Ethiopia), ii) the potential correlation between oocyst shedding pattern over time and Cryptosporidium species/genotype and iii) the potential correlation between oocyst shedding pattern over time and pathogenicity measured as diarrhoea or malnutrition severity. Detection of the parasite was accomplished by auramine-phenol fluorescence microscopy. Samples that tested positive by this method were reanalysed by PCR and Sanger sequencing to determine Cryptosporidium species and subtypes. Oocyst shedding was measured by conventional (microscopy counting) and mathematical (quantitative model) methods. Duration of oocysts shedding was found to last around four weeks, with C. hominis gp60 allele family Id having statistically significant longer shedding paters during middle and late infection stages. Another interesting finding is the estimation of a 10-fold decrease in Cryptosporidium DNA shedding quantity per week of infection. Overall, this study is relevant because estimating the intensity and duration of Cryptosporidium oocyst shedding may influence secondary transmission rates, suggesting that early treatment of cases could be a practical approach to minimize transmission dynamics of the parasite in endemic areas. However, there are some issues that need addressing or clarification.
Major issues 1. One of the main conclusions of the study is the proposal that Cryptosporidium oocyst shedding should be monitored as secondary treatment outcome. What is, in the opinion of the Authors the practical feasibility of this intervention in the routine practice of low-resource clinical settings? This would involve contacting the infected children through time and collecting several stool specimens. I wonder if adherence to this sample collection protocol would be an issue. 2. Methods, line 131: the Authors selected a PCR method specifically designed for the specific detection of C. hominis and C. parvum. Whereas it is true that both specie account for approximately 90% of the human cases of cryptosporidiosis characterised globally, the remaining 10% are caused by less frequent Cryptosporidium species including C. meleagridis, C. canis, C. felis, and C. ubiquitum, among others. Considering that these species have been described circulating in different human African populations (see for instance Squire and Ryan, Parasit Vectors 2017;10:195;Muadica et al. 2021;10:255;or Messa et al. Pathogens 2021;10:452, among many others), please comment on how this species detection limitation could have affected to the results obtained in the present study. 3. Line 207: it is unclear (and this is probably due to my lack of knowledge on this point), how the Authors can reach an estimation of the DNA quantity using mathematical modelling without a sample or a series sample to be used as reference/pattern. I think it would be useful to explain this in a way understandable for a non-technical audience.
Minor issues 1. Abstract, line 39: please provide a median and a range of the number of successive stool samples collected and analysed per children. 2. Methods, line 113-114: definition of diarrhoea is already included in line 167. Please avoid duplicating information. 3. Line 142: gp60, as other gene abbreviations, should be italicised. Please amend here and through the whole manuscript (e.g. lines 143, 145, 156, 160, 161, 186, 247, 250, 252, 290, 291, 323, 350, 351, 353, and 402). 4. Lines 402-406: cloning in bacteria could be another way of detecting (and differentiating) mixed infections involving different Cryptosporidium species/genotypes. Please add. 5. Line 411: please note that nitazoxanide is only prescribed in children older than 12 months of age. I would recommend modifying to 12-24 months.

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One of the main conclusions of the study is the proposal that Cryptosporidium oocyst shedding should be monitored as secondary treatment outcome. What is, in the opinion of the Authors the practical feasibility of this intervention in the routine practice of low-resource clinical settings? This would involve contacting the infected children through time and collecting several stool specimens. I wonder if adherence to this sample collection protocol would be an issue.
ANSWER: Here we are specifically thinking about research trials of nitazoxanide or new pharmaceutical or nutritional interventions against cryptosporidiosis, rather than a suggestion for a change in current clinical practice. We see that our use of the term "secondary treatment outcome" is misleading, and have clarified this in both Abstract:Conclusion and in the last paragraph of the Discussion, where we also added some context about outcome measures that were used in previous RCTs for cryptosporidiosis.

2.
Methods, line 131: the Authors selected a PCR method specifically designed for the specific detection of C. hominis and C. parvum. Whereas it is true that both specie account for approximately 90% of the human cases of cryptosporidiosis characterised globally, the remaining 10% are caused by less frequent Cryptosporidium species including C. meleagridis, C. canis, C. felis, and C. ubiquitum, among others. Considering that these species have been described circulating in different human ANSWER: We appreciate this important point and fully agree that we should not assume that other species may not be important in our study area, and that they are therefore important to look for. For this reason, we did not limit the analysis to C. parvum and C. hominis, but we do appreciate that this is not communicated clearly enough in the manuscript.
The distinction between the primary non-species specific qPCR and the C. parvum and C. hominis specific (lib13) PCR has now been made clearer (in Methods:Data collection) by labelling the Cryptosporidium qPCR assay as detecting "Cryptosporium spp. DNA", and by making it explicit that lib13 PCR was a follow-up assay after the initial round of qPCR, and, also, by including a separate line in Table 1 for "Cryptosporidium spp. other than C. parvum or C. hominis". We hope these modifications make it clear for the reader that we looked for all species, but that we did not find any "non-parvum non-hominis" Cryptosporidium detections in this study.
(For information, had there been any C non-hominis non-parvum detections, we would have proceeded with Cryptosporidium 18s rDNA (ssu) sequencing in order to determine the species. However, this was not necessary for the purposes of the current study)

3.
Line 207: it is unclear (and this is probably due to my lack of knowledge on this point), how the Authors can reach an estimation of the DNA quantity using mathematical modelling without a sample or a series sample to be used as reference/pattern. I think it would be useful to explain this in a way understandable for a non-technical audience.
ANSWER: Good point, we have tried to clarify the key idea behind the method (in Methods: Modelling Cryptosporidium shedding quantity over time), which is basically to make a "best fit" aggregate curve based on the drop in quantity (on the y axis) as a function of time (on the x axis) , and by using language (e.g., "plotted", "best fit", "aggregate curve") that we hope will prompt the reader to inspect the plots and appreciate the visual and fairly intuitive nature of the underlying statistical method.
We also added some detail on the qPCR assay to Methods:Data collection, including a specific mention that we used standard curves of serially diluted known quantities of Cryptosporidium DNA to calibrate the qPCR.

1.
Abstract, line 39: please provide a median and a range of the number of successive stool samples collected and analysed per children.
ANSWER: This has now been added to Abstract:Results and Results:1 st paragraph.

2.
Methods, line 113-114: definition of diarrhoea is already included in line 167. Please avoid duplicating information.
ANSWER: The full definition is now provided at first mention in Methods:Study design and participants, and deleted from Methods:Definitions.
ANSWER: Thank you for pointing this out, we have corrected all gene mentions to small letters and italics, including (i.e., gp60 and lib13).

4.
Lines 402-406: cloning in bacteria could be another way of detecting (and differentiating) mixed infections involving different Cryptosporidium species/genotypes. Please add.
ANSWER: Good point, we have now mentioned both cloning in a vector, and a new and promising bioinformatics tool, that can be useful for elucidating mixed gp60 infections, in the second last paragraph of Discussion.

5.
Line 411: please note that nitazoxanide is only prescribed in children older than 12 months of age. I would recommend modifying to 12-24 months. ANSWER: Our remark was not intended to apply to just the use of nitazoxanide, but also to new therapeutics that may be developed in the future, but we appreciate that this was not clear. Furthermore, when it comes to therapeutic interventions, as our study is an observational study, we hope that it can inform further research (e.g., to provide useful data justifying the evaluation of shedding duration as a trial outcome) rather than a direct change in clinical practice. We have therefore rephrased the sentence (Discussion:last paragraph) to make this distinction clear to the reader.
On nitazoxanide specifically, the reviewer is here reminding us of the important fact that nitazoxanide is not currently FDA-approved for children 12 months or older. We think this is somewhat regrettable, as there is available clinical evidence of safety in trials that also included children younger than 12 months. Furthermore, the ongoing NICE-GUT trial in Australia is aiming to provide additional safety data in infants older than 3 months. We however acknowledge that this important context was missing from the manuscript, and it has now been provided in the Introduction.

RESPONSE TO EDITOR'S COMMENTS:
"In addition to addressing the reviewer comments, it will be useful to strengthen the introduction section. The significance and value of this study needs to be discussed by comparing what is known from other epidemiological studies such as GEMS-1A, Crypto-POC etc. Also, it will be useful for the reader if more information is provided in the introduction section on prevalence of Cryptosporidium species, C. hominis vs C. parvum (lines 60-62). Sensitivity of oocyst detection (detection limits) by qPCR also needs to be discussed in the results and discussion section. Moreover, the quality of the figures provided can be improved and high-resolution figures will be required." RESPONSE: The Introduction section has been expanded with some information on the molecular epidemiology of cryptosporidiosis in Africa, and some framing information referring to the invaluable information on risk association, morbidity, and mortality from the GEMS-1A and GBD studies (Introduction:1 st paragraph).
We also provided more specific information on previous studies that have investigated shedding duration, to allow for comparison with the MAL-ED study on postdiarrhoeal shedding in a community setting, and to point out some features that distinguish these previous studies from the current study (Introduction:2 nd paragraph).
Further details on the lowest reliable detection limit has now been added to Results:1 st paragraph and we mention the possible impact of the different target in our qPCR assay compared with the target that was used in the MAL-ED study on postdiarrhoeal shedding (Discussion:1 st paragraph)